While many small proteins can refold spontaneously in vitro, the identification of the folding pathway, and the detection and structural characterization of folding intermediates have been difficult. Recently, attention has turned to the molten globule state and other nonnative equilibrium states of proteins which are thought to be models for kinetic intermediates in protein folding. The molten globule state of apomyoglobin can be populated at equilibrium at reduced Ph as demonstrated by a number of optical and hydrodynamic methods, and has recently been shown to represent a kinetic intermediate on the myoglobin folding pathway. Amide protection studies have identified that only the A, G, and H helices of myoglobin are protected in the molten globule state, suggesting that a native-like substructure involving those regions may persist. Fragments of staphylococcal nuclease have been produced which also have properties similar to those of the molten globule, i.e. a somewhat compact structure with some secondary structure but without a defined tertiary structure. Recently we have developed a chemical cleavage method where an EDTA-Fe based reagent (EPD) can be attached to a protein via a cysteine side chain. The addition of ascorbate generates hydroxyl radicals at the iron center which diffuse and cleave the polypeptide backbone in a region close to the cysteine attachment site at residues accessible to solvent. The observed cleavage sites can be mapped by amino acid sequencing. The cleavage is dependent on protein conformation. We propose to characterize the molten globule state of apomyoglobin and staphylococcal nuclease fragment structures using this newly developed chemical cleave technique. We will prepare a number of cysteine variants of these proteins and characterize the cleavage patterns observed in the native and molten globule states.